CN108060192A - For improving the fermentation medium of meleumycin fermentation level and feed process - Google Patents
For improving the fermentation medium of meleumycin fermentation level and feed process Download PDFInfo
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- CN108060192A CN108060192A CN201610986026.7A CN201610986026A CN108060192A CN 108060192 A CN108060192 A CN 108060192A CN 201610986026 A CN201610986026 A CN 201610986026A CN 108060192 A CN108060192 A CN 108060192A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/44—Preparation of O-glycosides, e.g. glucosides
- C12P19/60—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin
- C12P19/62—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin the hetero ring having eight or more ring members and only oxygen as ring hetero atoms, e.g. erythromycin, spiramycin, nystatin
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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Abstract
The present invention provides a kind of fermentation medium for being used to improve meleumycin fermentation level, including glucose 1.5 3.0%, analysis for soybean powder 2 5%, dusty yeast 0.1 0.3%, fish meal 0.1 0.5%, potassium dihydrogen phosphate 0.05 0.2%, magnesium sulfate 0.05 0.2% and oil 1 10%.The present invention also provides a kind of methods for improving meleumycin fermentation level, utilize above-mentioned culture medium, using Streptomyces Macrofaciens as fermenting microbe, when the pH value of zymotic fluid is rebounded by minimum point to 6.0, by adding glucose solution, so as to improve the ratio of the fermentation level of meleumycin and kitasamycin.The present invention is simple and practicable, and using the method for the present invention fermenting and producing meleumycin, content is up to 3400 4000mg/L, and weight ratio shared by midecamycin a1 is about 58.38 59.75%, and weight ratio is up to 24.72 29.72% shared by kitasamycin A6；And the tank production of meleumycin is do not add glucose solution control about 1.2 times or more after fermenting, A6 components can improve about more than 25%.
Description
Technical field
The present invention relates to microbial fermentation and biopharmaceutical technologies, specifically, are related to a kind of white for improving wheat
The fermentation medium and feed process of mycin fermentation level.
Background technology
Meleumycin (Meleumycin, MLM) is isolated in succession from Sichuan, Soils of Guangdong by China's researcher
Streptomycete (S.mycarofaciens10204 and 1748), it is fermented refinement and obtain.Meleumycin to staphylococcus aureus,
Erythromycin-sensitive bacterium in staphylococcus epidermis, micrococcus scarlatinae and pneumococcus has preferable antibacterial action, to tiny
Other common clinical pathogenic bacteria such as streptococcus and Diplococcus gonorrhoeae have certain antibacterial activity.
Requirement of 2005 editions pharmacopeia to medecamycin is must not to be less than 35.0% containing midecamycin a1.With changing for pharmacopeia
Version, the requirement to meleumycin further improve.Midecamycin a1 should not be low in the two regulation meleumycins of version pharmacopeia in 2010
In 48%, kitasamycin A6 should be not less than 12%, and A1, A2, A4, A6, A8 summation should be not less than 70%.However, at present in scientific research
In actual production, kitasamycin A6 proportions caused by the fermentation of meleumycin producing strains are relatively low, and there are unqualified for product
Risk it is larger.
Find that meleumycin fermentation thalli is higher to the demand of oxygen, causes to ferment when cell concentration is excessively high in experimentation
Process oxygen is insufficient, influences yield.If cell concentration is too low, production meleumycin speed is slower, and fermentation period is long, thalline
Preferable effect is equally not achieved in aging.Therefore the dissolved oxygen for improving zymotic fluid is to solve the above problems, and that improves fermentation level has
Effect approach.Meanwhile requirement of the meleumycin during the fermentation to being metabolized pH is higher, pH is in 5-6 in metabolic process, A6 groups
The growth divided is optimal.Once metabolic process pH is raised, dissolved oxygen reduces speed and can speed, and the growth of A6 components also can slow down or even stop
Only, while strain can be easier self-dissolving and zymotic fluid is caused to blister, and not only influence fermentation yield and have an effect on refinement press filtration.It attempts to mend acid
The rise of pH can only be briefly controlled, pH understands rapid increase again quickly, while regulates and controls fermentating metabolism environment by mending acid, to bacterium
The stimulation of kind is big, can not solve the problems, such as that the growth of A6 components slows down and even stagnate.
The content of the invention
Present invention seek to address that in existing meleumycin fermentation manufacturing technique occur metabolism pH rise simultaneously with dissolved oxygen not
Foot, the problem of ultimately resulting in kitasamycin stagflation and thalline self-dissolving, provide a kind of for improving meleumycin fermentation level
Feed process.
It is a further object of the present invention to provide a kind of for improving the fermentation medium of meleumycin fermentation level.
In order to realize the object of the invention, present invention firstly provides a kind of meleumycin fermentation culture medium, the culture mediums
In contain following each component：Glucose 1.5-3.0%, analysis for soybean powder 2-5%, dusty yeast 0.1-0.3%, fish meal 0.1-0.5%, phosphorus
Acid dihydride potassium 0.05-0.2%, magnesium sulfate 0.05-0.2% and corn oil 1-10%.
In above-mentioned culture medium, glucose is carbon source, in addition, vegetable oil, molasses, sucrose, dextrin, maltodextrin, starch etc.
Also carbon source can be used as；Analysis for soybean powder, dusty yeast and fish meal are nitrogen source, in addition, bean cake powder, peptone, groundnut meal etc. also can conducts
Nitrogen source；Inorganic salts can also include dipotassium hydrogen phosphate, sodium chloride etc..The culture medium being made of more than carbon source, nitrogen source and inorganic salts
Belong to the scope of the present invention.
Preferably, following each component is contained in the culture medium：Glucose 2%, analysis for soybean powder 4%, dusty yeast 0.2%, fish
Powder 0.4%, potassium dihydrogen phosphate 0.1%, magnesium sulfate 0.1% and corn oil 8%.
The present invention provides a kind of method for improving meleumycin fermentation level, is cultivated using above-mentioned meleumycin fermentation
Base, using Streptomyces Macrofaciens (Streptomyces mycarofaciens) as fermenting microbe, when the pH value of zymotic fluid is by most
When low spot is rebounded to 6.0, by adding glucose solution, fermentation is effectively had adjusted while the dissolved oxygen amount in improving zymotic fluid
Metabolic process material liquid pH (improves A6 ratios so as to improve the fermentation level of meleumycin and improve kitasamycin constituent content
Example).
In preceding method, the concentration of glucose solution is added as 5%-10%, and dosage is the 2%-10% of fermentating liquid volume.
Preferably, the dosage of glucose solution is the 4%-7% of fermentating liquid volume.It is highly preferred that the temperature for the glucose solution added
It spends for 26-28 DEG C.
In preceding method, the additional way of glucose solution is added for disposable flowing, and stream rate of acceleration is 200ml/h.
Preceding method comprises the following steps：
1) preparation of seed liquor：Viable bacteria amount is 1 × 10 in seed liquor obtained7-1×108CFU/mL；
2) fermenting and producing of meleumycin：By the inoculum concentration (preferably 10%) of 8-10%, seed liquor prepared by step 1)
Access equipped with the fermentation of 30L meleumycins with the volume of culture medium be 50L fermentation tank in, in 26-28 DEG C, speed of agitator 100-
Fermented and cultured is carried out under conditions of 300rpm, air mass flow 600L/h, when the pH value of zymotic fluid is rebounded by minimum point to 6.0,
Glucose solution is added, fermentation period is 5-8 days.
Present invention fermentation bacterial strain uses therefor is S.mycarofaciens var.Sichuansis.It is other to can be used for wheat white mould
The bacterial strain of plain fermenting and producing falls within the scope of the present invention.
Wherein, the preparation method of the seed liquor is as follows：Streptomyces Macrofaciens are accessed into Gause I solid slope culture
Base is cultivated 5 days in 28 DEG C；Then by shaking flask of the strain access equipped with liquid seed culture medium on slant medium, liquid is filled
100mL/750mL is measured, in 28 DEG C, speed of agitator 220rpm, to get seed liquor when shaken cultivation 30 is small.
The formula of the liquid seed culture medium is：Glucose 1-2%, starch 1-3%, analysis for soybean powder 2-4%, biphosphate
Potassium 0.01-0.05%, ammonium sulfate 0.2-0.5% and calcium carbonate 0.1-0.3%.
Using the meleumycin of the method for the present invention fermenting and producing, content accounts for wheat up to 3400-4000mg/L, midecamycin a1
The weight ratio of albomycin is about 58.38-59.75%, and kitasamycin A6 accounts for the weight ratio of meleumycin up to 24.72-
29.72%；And the tank production of meleumycin is do not add glucose solution control about 1.2 times or more after fermenting, A6 components can
Improve about more than 25%.The final finished quality that rises to of zymotic fluid quality is laid a good foundation, and is allowed to meet and be wanted higher than pharmacopeia
It asks, reduces the risk of substandard product generation, be of great significance to actual production.
Specific embodiment
Following embodiment is not limited to the scope of the present invention for illustrating the present invention.Unless otherwise specified, embodiment
In the conventional means that are well known to those skilled in the art of used technological means, raw materials used is commercial goods.
Following embodiment bacterial strain uses therefor is S.mycarofaciens var.Sichuansis.
The percentage sign " % " arrived involved in the present invention, if not specified, refers to mass percent；But the percentage of solution
Than unless otherwise specified, referring to the grams containing solute in 100mL solution.
Embodiment 1 is used to improve the feed process of meleumycin fermentation level
Meleumycin fermentation culture medium provided in this embodiment contains following each component：Glucose 2%, analysis for soybean powder 4%,
Dusty yeast 0.2%, fish meal 0.4%, potassium dihydrogen phosphate 0.1%, magnesium sulfate 0.1% and corn oil 8%.Fermentation process is as follows：
1st, the preparation of seed liquor：Streptomyces Macrofaciens are accessed into Gause I solid slope culture medium, 5 are cultivated in 28 DEG C
My god；Then the strain access on slant medium is equipped in the shaking flask of liquid seed culture medium, liquid amount 100mL/750mL,
In 28 DEG C, speed of agitator 220rpm, to get seed liquor when shaken cultivation 30 is small.In gained seed liquor viable bacteria amount be about 6 ×
107CFU/mL。
The formula of the liquid seed culture medium is：Glucose 1%, starch 2%, analysis for soybean powder 3%, potassium dihydrogen phosphate
0.03%th, ammonium sulfate 0.3% and calcium carbonate 0.2%.
2nd, the fermenting and producing of meleumycin：By 10% inoculum concentration, seed liquor access prepared by step 1 is equipped with 30L wheats
Albomycin fermentation with the volume of culture medium be 50L fermentation tank in, in 28 DEG C, speed of agitator 200rpm, air mass flow 600L/h
Under conditions of carry out fermented and cultured, when the pH value of zymotic fluid is rebounded by minimum point to 6.0, concentration 6% is added in disposable flowing
Glucose solution 1.5L, stream rate of acceleration be 200ml/h, fermentation period be 6 days.Fermentation ends, put tank, and zymotic fluid pH is
5.68, put tank volume 28.5L.
With each component of high performance liquid chromatography detection meleumycin, wheat white mould is measured using antibiotic-microbial assay
Cellulose content, detection method is referring to 2010 editions pharmacopeia.
Meleumycin content is 4030mg/L in the present embodiment, and the weight ratio that midecamycin a1 accounts for meleumycin is
The weight ratio that 59.78%, kitasamycin A6 account for meleumycin is 29.72%.Control group (its without adding glucose solution
Its processing step and dispensing are same as above), zymotic fluid meleumycin content 3100mg/L measures A1 in meleumycin after fermentation
The weight ratio that weight ratio is 56.45%, A6 is 19.58%, puts tank volume 27L.Add the wheat for generation of fermenting after glucose solution
Albomycin puts tank volume and is higher by about 5% than control group.Total amount is not add the control group generation meleumycin of glucose solution
About 1.37 times of tank production.
Embodiment 2 is used to improve the feed process of meleumycin fermentation level
Meleumycin fermentation culture medium provided in this embodiment contains following each component：Glucose 1.5%, analysis for soybean powder
5%th, dusty yeast 0.1%, fish meal 0.5%, potassium dihydrogen phosphate 0.05%, magnesium sulfate 0.2% and corn oil 1%.
Fermentation process is with described in embodiment 1, and when the pH value of zymotic fluid is rebounded by minimum point to 6.0, disposable flowing is mended
Add 3% glucose solution 3L of concentration, stream rate of acceleration is 200ml/h, and fermentation period is 5 days.Fermentation ends put tank, zymotic fluid pH
For 5.82, tank volume 30L is put.
Meleumycin content is 3410mg/L in the present embodiment, and the weight ratio that midecamycin a1 accounts for meleumycin is
The weight ratio that 58.35%, kitasamycin A6 account for meleumycin is 25.46%.The wheat for adding generation of fermenting after glucose solution is white
Mycin puts tank volume and is higher by about 10% than control group.Total amount is not add the control group generation meleumycin tank of glucose solution
About 1.21 times of production.
Embodiment 3 is used to improve the feed process of meleumycin fermentation level
Meleumycin fermentation culture medium provided in this embodiment contains following each component：Glucose 3%, analysis for soybean powder 2%,
Dusty yeast 0.3%, fish meal 0.1%, potassium dihydrogen phosphate 0.2%, magnesium sulfate 0.05% and corn oil 10%.
Fermentation process is with described in embodiment 1, and when the pH value of zymotic fluid is rebounded by minimum point to 6.0, disposable flowing is mended
Add 15% glucose solution 0.6L of concentration, stream rate of acceleration is 200ml/h, and fermentation period is 8 days.Fermentation ends put tank, zymotic fluid
PH is 5.58, puts tank volume 27.6L.
Meleumycin content is 3720mg/L in the present embodiment, and the weight ratio that midecamycin a1 accounts for meleumycin is
The weight ratio that 58.78%, kitasamycin A6 account for meleumycin is 24.72%.The wheat for adding generation of fermenting after glucose solution is white
Mycin puts tank volume and is higher by about 2% than control group.Total amount is not add the control group generation meleumycin tank of glucose solution
About 1.22 times of production.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (8)
1. meleumycin fermentation culture medium, which is characterized in that contain following each component in the culture medium：Glucose 1.5-
3.0%th, analysis for soybean powder 2-5%, dusty yeast 0.1-0.3%, fish meal 0.1-0.5%, potassium dihydrogen phosphate 0.05-0.2%, magnesium sulfate
0.05-0.2% and corn oil 1-10%.
2. culture medium according to claim 1, which is characterized in that contain following each component in the culture medium：Glucose
2%th, analysis for soybean powder 4%, dusty yeast 0.2%, fish meal 0.4%, potassium dihydrogen phosphate 0.1%, magnesium sulfate 0.1% and corn oil 8%.
A kind of 3. method for improving meleumycin fermentation level, which is characterized in that using the culture medium described in claim 1 or 2,
Using Streptomyces Macrofaciens (Streptomyces mycarofaciens) as fermenting microbe, when the pH value of zymotic fluid is by minimum point
When rebound is to 6.0, by adding glucose solution, so as to improve the fermentation level of meleumycin and improve kitasamycin group
Divide content.
4. according to the method described in claim 3, it is characterized in that, add the concentration of glucose solution as 5%-10%, dosage
For the 2%-10% of fermentating liquid volume；Preferably, dosage is the 4%-7% of fermentating liquid volume；It is highly preferred that the grape added
The temperature of sugar juice is 26-28 DEG C.
5. according to the method described in claim 4, it is characterized in that, the additional way of glucose solution is mended for disposable flowing
Add, stream rate of acceleration is 200ml/h.
6. according to claim 3-5 any one of them methods, which is characterized in that comprise the following steps：
1) preparation of seed liquor：Viable bacteria amount is 1 × 10 in seed liquor obtained7-1×108CFU/mL；2) fermentation of meleumycin
Production：By the inoculum concentration of 8-10%, appearance of the seed liquor access equipped with 30L meleumycin fermentation culture mediums prepared by step 1)
Product is in the fermentation tank of 50L, is fermented under conditions of 26-28 DEG C, speed of agitator 100-300rpm, air mass flow 600L/h
Culture when the pH value of zymotic fluid is rebounded by minimum point to 6.0, adds glucose solution, and fermentation period is 5-8 days.
7. according to claim 3-6 any one of them methods, which is characterized in that fermentation bacterial strain uses therefor be
S.mycarofaciens var.Sichuansis。
8. according to claim 3-7 any one of them methods, which is characterized in that the preparation method of the step 1) seed liquor is such as
Under：Streptomyces Macrofaciens are accessed into Gause I solid slope culture medium, are cultivated 5 days in 28 DEG C；It then will be on slant medium
Strain access equipped with liquid seed culture medium shaking flask in, liquid amount 100mL/750mL, in 28 DEG C, speed of agitator 220rpm,
To get seed liquor when shaken cultivation 30 is small；
Wherein, the formula of the liquid seed culture medium is：Glucose 1-2%, starch 1-3%, analysis for soybean powder 2-4%, di(2-ethylhexyl)phosphate
Hydrogen potassium 0.01-0.05%, ammonium sulfate 0.2-0.5% and calcium carbonate 0.1-0.3%.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111961700A (en) * | 2019-04-16 | 2020-11-20 | 天方药业有限公司 | Fermentation method of kitasamycin |
| CN115896217A (en) * | 2022-12-16 | 2023-04-04 | 内蒙古中牧生物药业有限公司 | Feed supplementing process for improving fermentation level of tulathromycin |
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| CN102676617A (en) * | 2011-03-17 | 2012-09-19 | 河南天方药业股份有限公司 | Preparation method for meleumycin |
| CN103103236A (en) * | 2011-11-14 | 2013-05-15 | 华远医药研究院有限公司 | New process for preparing meleumycin |
| CN104109702A (en) * | 2014-06-30 | 2014-10-22 | 北大医药重庆大新药业股份有限公司 | Fermentation culture medium for meleumycin |
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| CN102676617A (en) * | 2011-03-17 | 2012-09-19 | 河南天方药业股份有限公司 | Preparation method for meleumycin |
| CN103103236A (en) * | 2011-11-14 | 2013-05-15 | 华远医药研究院有限公司 | New process for preparing meleumycin |
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Cited By (3)
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|---|---|---|---|---|
| CN111961700A (en) * | 2019-04-16 | 2020-11-20 | 天方药业有限公司 | Fermentation method of kitasamycin |
| CN111961700B (en) * | 2019-04-16 | 2023-04-28 | 天方药业有限公司 | Fermentation method of kitasamycin |
| CN115896217A (en) * | 2022-12-16 | 2023-04-04 | 内蒙古中牧生物药业有限公司 | Feed supplementing process for improving fermentation level of tulathromycin |
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Effective date of registration: 20230612 Address after: 3007, Hengqin international financial center building, No. 58, Huajin street, Hengqin new area, Zhuhai, Guangdong 519031 Patentee after: New founder holdings development Co.,Ltd. Patentee after: CHONGQING DAXIN PHARMACEUTICAL Co.,Ltd. Patentee after: Peking University Medical Management Co.,Ltd. Address before: 100871 8th floor, founder building, 298 Chengfu Road, Haidian District, Beijing Patentee before: PEKING UNIVERSITY FOUNDER GROUP Co.,Ltd. Patentee before: CHONGQING DAXIN PHARMACEUTICAL Co.,Ltd. Patentee before: PKU HEALTHCARE INDUSTRY Group |